Reduction products of everninomicins and methods for their manufacture

ABSTRACT

Everninomicin antibiotics having a nitro function, e.g. everninomicin B, everninomicin C, and everninomicin D, upon treatment with aluminum amalgam in aqueous alkanol are converted to a mixture of at least two reduction products having antibacterial activity, said reduction products being an everninomicin having a nitroso function and an everninomicin having a hydroxylamino function. The nitrosoeverninomicins are also prepared by treating the corresponding hydroxylaminoeverninomicin with an oxidizing reagent selected from the group consisting of aerial oxidation in alkaline solution, and an alkali metal hypobromite in an aprotic solvent. The everninomicin reduction products and salts thereof are described as well as nitrone derivatives of the hydroxylaminoeverninomicins and salts thereof, all of which possess antibacterial activity. A preferred compound is hydroxylaminoeverninomicin D, particularly the sodium salt thereof.

United States Patent 1191 Ganguly et al.

[ Oct. 28, 1975 REDUCTION PRODUCTS OF EVERNINOMICINS AND METHODS FOR Primary Examiner-Johnnie Brown THEI MANUFACTURE Assistant ExaminerCary Owens [75] Inventors: Ashit K. Ganguly, Upper Montclair; Attorney Agent or FlrmMary Kmg Olga Sari-e, Verona, both of NJ. [57] ABSTRACT [73] Asslgneei Schel'ing Corporation, Kenilwonh: Everninomicin antibiotics having a nitro function, e.g. everninomicin B, everninomicin C, and everninomicin 22 i Oct 31, 1973 D, upon treatment with aluminum amalgam in aqueous alkanol are converted to a mixture of at least two PP 411,548 reduction products having antibacterial activity, said Related s Application Data reduction products being an everninomicin having a [63] Continuatiomimpan of Ser No 377 344 Jul 9 nitroso function and an everninomicin having a hy- 1973 abandoned which is droxylamino function. The nitrosoeverninomicins are Sen 1110. 315,263Dec 14 1972 abandoned p also prepared by treating the corresponding hydroxylaminoeverninomicin with an oxidizing reagent se- 52 vs. C! 260/210 AB; 424/180 lected from the group consisting f aerial Oxidation in 51 1111.01. c070 3/00 alkaline Solution, and an alkali metal hypobromite in 58 Field of Search 260/210 R, 210 AB an aprotic solvent The eveminomicin reduction P ucts and salts thereof are described as well as nitrone References Cited derivatives of the hydroxylaminoeverninomicins and UNITED STATES PATENTS salts thereof, all of which possess antibacterial activity.

A preferred compound is hydroxylaminoeverninomii: 2529 5 cin D, particularly the sodium salt thereof. 3,674,773 7 1972 Kurath 260/210 E 16 l im Drawing Figures 51300 aopo 00 1590 1290 1190 1090 900 sec 2 V 80 l-Ll 5 6O F E 2 m 2 E 20- O l l I l l I I I WAVELENGTH M ICRONS) INFRARED SPECTRUM OF HYDROXYLAMINOEVERNINOMICIN D US. Patent O6L28, 1975 Sheet1of4 3,915,956

3800 30 00 2o oo :spo 1290 mp0 I090 990 850 TRANSMITTANCE ("/o) 0 i 1 5 7 25 lb I I2 WAVELENGTH (MICRONS) INFRARED SPECTRUM OF HYDROXYLAMINOEVERNINOMlClN D FIG. 1

l l l I l l I 8.0 7.0 6.0 5.0 4.0 3.0 2.0 LO 0 NUCLEAR MAGNETIC RESONANCE SPECTRUM OF HYDROXYLAMINOEVERNINOMICIN D FIG. 2

US. Patent Oct. 28, 1975 Sheet20f4 3,915,956

4000 3000 2000 I500 CIVFl I200 ||00 I000 900 850 O 3 5 5 3 [0 I I l2 WAVELENGTH (MICRONS) INFRARED SPECTRUM OF NITROSOEVERNINOMIC'N D FIG. 3

I l I l I I I 8.0 7.0 6.0 5,0 4.0 3.0 2.0 1.0 0

NUCLEAR MAGNETIC RESONANCE SPECTRUM 0F NITROSOEVERNINOMICIN D FIG. 4

U.S. Patent TRANSMITTANCE ("/o) TRANSMITTANCE (%I TRANSMITTANCE ("/o) Sheet 3 of4 3,915,956

Oct. 28, 1975 I200 I I00 I000 5 5 6 5 :5 WAVELENGTH (MICRONS) INFRARED SPECTRUM OF HYDROXYLAMINOEVERNINOMICIN D BENZYL NITRONE FIG. 5

I200 H00 I000 900 '3 A 5 3 5 WAVELENGTH (MICRONS) INFRARED SPECTRUM OF HYDROXYLAMINOEVERNINOMICIN D FURFURYL N ITRONE FIG. 6

I200 I I00 I000 900 I0. I I I2 I3 5 4 5 6 7 9 WAVELENGTH (MICRONS) INFRARED SPECTRUM 0F HYDROXYLAMINOEVERNINOMICIN D HEPTYL NITRONE FIG. 7

U.S. Patent 0a. 28, 1975 Sheet4 0f4 3,915,956

I200 II00 O O O O 8 6 4 2 INFRARED SPECTRUM OF EVERNINOMICIN C WAVELENGTH (M ICRONSI FIG. 6

I500 I200 I100 I000 900 O O O 0 8 6 4 2 INFRARED SPECTRUM OF HYDROXYLAMINOEVERNINOMICIN C WAVELENGTH (MICRONSI FIG. 9

I200 IIOO I000 O O O O 8 6 4 2 INFRARED SPECTRUM 0F HYDROXYLAMINOEVERNINOMICIN B WAVELENGTH (MICRONS) FIG. 10

REDUCTION PRODUCTS OF EVERNINOMICINS AND METHODS FOR THEIR MANUFACTURE CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of copending application U.S. Ser. No. 377,344, filed July 9, 1973, in turn a continuation-in-part of application U.S. Ser. No. 3 15,263, filed Dec. 14, 1972, both now abandoned.

FIELD OF INVENTION This process relates to novel compositions of matter and to processes for their preparation.

More specifically, this invention relates to everninomicins having a nitroso or a hydroxylamino function and derivatives thereof which exhibit antibacterial properties, and to methods for their preparation.

In particular, this invention relates to antibacterials selected from the group consisting of nitrosoeverninomicin B, nitrosoeverninomicin C, nitrosoeverninomicin D, hydroxylaminoeverninomicin B, hydroxylaminoeverninomicin C, hydroxylaminoeverninomicin D, nitrone derivatives of said hydroxylaminoeverninomicins, and pharmaceutically acceptable salts of the foregoing. This invention also relates to the processes for preparing the foregoing antibacterials and derivatives thereof and to intermediates produced thereby.

PRIOR ART A mixture of everninomicin antibiotics, including everninomicin B, everninomicin C, and everninomicin D are produced when Micromonospora carbanacea var. carbonacea and a variant thereof, Micromonospora carbonacea var. aurantiacea are subjected to submerged aerobic fermentation under conditions described in US. Pat. No. 3,499,078.

Described in the art are the physical characteristics of everninomicin B and everninomicin D, each of which contain a nitro function. We have now determined that eveminomicin C also contains a nitro function.

By our invention we have discovered that when an everninomicin antibiotic having a nitro function, e.g. everninomicin B, everninomicin C, and everninomicin D, is treated with aluminum amalgam in an aqueous alkanol, there is produced a mixture of at least two reduction products having antibacterial activity, said reduction products being the corresponding eveminomicin wherein said nitro group has been converted to a nitroso or a hydroxylamino function, e.g. nitrosoeverninomicin B and hydroxylaminoeveminomicin B, nitrosoeverninomicin C and hydroxylaminoeverninomicin C, and nitrosoeverninomicin D and hydroxylaminoeverninomicin D, respectively.

It is apparent that each of the aforementioned hydroxylaminoeveminomicin antibacterials can also be prepared by reducing the corresponding nitrosoeverninomicins by methods known to reduce nitroso functions to hydroxylamino functions.

By our invention, we. have also discovered that the nitrosoeverninomicin antibacterials are preferably prepared by oxidizing the corresponding hydroxylaminoeverninomicin antibacterial by means selected from the group consisting of aerial oxidation in alkaline solution, and an alkali metal hypobromite in an aprotic solvent.

We have further determined the structure of everninomicin B, everninomicin C, and everninomicin D and reduction products thereof as disclosed herembelow.

SUMMARY OF INVENTION In one process of the invention sought to be patented, an everninomicin antibiotic having a nitro group is treated with aluminum amalgam whereby is produced a mixture of antibacterials comprising at least two everninomicin reduction products having antibacterial activity, said mixture comprising a member selected from the group consisting of a nitrosoeverninomicin and a hydroxylaminoeveminomicin. When the nitro-containing everninomicin antibiotic starting compound of this process is a member selected from the group consisting of everninomicin B, everninomicin C, and everninomicin D, there is produced an antibacterial mixture comprising a member selected from the group consisting of nitrosoeverninomicin B and hydroxylaminoeverninomicin B, nitrosoeverninomicin C and hydroxylaminoeverninomicin C, and nitrosoeverninomicin D and hydroxylaminioeveminomicin D, respectively. A preferred species of this process is that wherein everninomicin D is the starting antibiotic, wherein said aluminum amalgam is an amalgam of Reynolds Wrap aluminum foil, and wherein said process is carried out in aqueous lower alkanol, whereby is produced a mixture of antibiotics comprising a member selected from the group consisting of nitrosoeverninomicin D and hydroxylaminoeveminomicin D.

In another process aspect of this invention, a hydroxylaminoeverninomicin antibacterial is treated with an oxidizing reagent selected from the group consisting of aerial oxidation in an alkaline solution, and an alkali metal hypobromite in an aprotic solvent whereby is produced a nitrosoeverninomicin having antibacterial activity. When the starting compound of this process is a member selected from the group consisting of hydroxylaminoeveminomicin B, hydroxylaminoeverninomicin C and hydroxylaminoeverninomicin D, there is produced a nitrosoeverninomicin selected from the group consisting of nitrosoeverninomicin B, nitrosoeverninomicin C, and nitrosoeverninomicin D.

In its composition of matter aspect, the invention sought to be patented resides in the concept of an everninomicin derivative selected from the group consisting of nitrosoevernimoicin B, nitrosoeverninomicin C, nitrosoeverninomicin D, hydroxylaminoeverninomicin B, hydroxylaminoeveminomicin C, hydroxylaminoeverninomicin D, nitrone derivatives of the foregoing hydroxylaminoeveminomicins, and pharmaceutically acceptable salts of the foregoing. A preferred species of this aspect of our invention is hydroxylaminoeverninomicin D and nitrone derivatives thereof and the pharmaceutically acceptable salts of the foregoing which are more rapidly absorbed than, and have antibacterial activity comparable to that of everninomicin D. Of particular value as an antibacterial is hydroxylaminoeverninomicin D sodium salt.

GENERAL DESCRIPTION OF THE INVENTION The Reduction Process:

Our process whereby an everninomicin antibiotic having a nitro group is treated with aluminum amalgam to produce a mixture comprising at least two antibacterials selected from the group consisting of a nitroso- 3 everninomicin and a hydroxylaminoeverninomicin, is usually carried out at room temperature in an aqueous alkanol (preferably aqueous ethanol).

The aluminum amalgam for use in our process is prepared from aluminum and mercuric chloride in alkali according to known procedures. We have found that our process proceeds at a faster rate and produces higher yields of hydroxylaminoeverninomicin when the aluminum amalgam is freshly prepared just prior touse in our reduction process and when the amalgam is prepared with aluminum foil which is comprised essentially of from about 98% to about 99.5% aluminum usually together with trace quantities of at least one member selected from the group consisting of copper, silicone and iron, particularly when prepared with the aluminum foil sold commercially under the trade name Reynolds Wrap which usually comprises from about at least 99.35% aluminum and about 0.12% copper. When preparing the amalgam, we usually cut the aluminum foil in 0.5 cm. squares which are then folded at random into ball-like shapes prior to treatment with alkali and mercuric chloride.

The starting compounds for our process are everninomicin antibiotics having a nitro group, particularly everninomicin B, everninomicin C, and everninomicin D which are known antibiotics produced by the aerobic fermentation of Micromonospora carbonacea var. carbonacea and a variant thereof Micromonospora carbonacea var. aurantia according to procedures known in the art such as described in US. Pat. No. 3,499,078. The isolation and purification and everninomicins B and D are carried out essentially as described in the art and the isolation, purification and characterization of everninomicin C are carried out in a similar manner as described herein.

Of antibiotics everninomicins B, C, and D, everninomicin D is most readily available and is a preferred starting compound for our reduction process since there is produced therefrom good yields of hydroxylaminoeverninomicin D, a preferred compound .of the composition of matter aspect of our invention. Everninomicin D is a compound of following structural formula I wherein X is nitro, i.e. X is By our reduction process, the nitro group in the evernitrose sugar group present in everninomicin D, as defined by above structural formula I, is reduced to produce a mixture of products comprising at least two novel compounds having antibacterial activity; in one, the nitro group has been reduced to a nitroso function (i.e. to a compound of formula I wherein Y is hydrogen, X is --N=O and Z is to produce nitrosoeverniomicin D; in another, the nitro group has been reduced to a hydroxylamino function (i.e. to a compound of structural formula I wherein X is Y is hydrogen and Z is CH/ CH3 ocH to produce hydroxylaminoeverninomicin D.

Other starting intermediates of our process, i.e. everninomicin B and C, also possess the evemitrose sugar group, the nitro function of which is reduced upon treatment with aluminum amalgam in aqueous alkanol to produce a mixture of antibacterials comprising at least two having a structure corresponding to that of the starting everninomicin, but with the nitro group of the everninomicin starting compound replaced by a ni- 5 troso or hydroxylamino function. The everninomicin B derivatives are compounds of formula I wherein Y is hydroxyl and Z is Y is hydrogen, and is 50 CH cn, CH 3 CH \OCHa OCH;

OCH3 Cl 0 z OCH no i:\ CH 3 3 O 0 3 c 3 o c3 0 ca o o 0H 9C8 H o ORA on 3 X CH whereas everninomicin C derivatives are compounds of formula I wherein Y is hydrogen and Z is hydrogen.

When carrying out our reduction process, it is advantageous to repurify the starting everninomicin antibiotic just prior to treatment thereof with aluminum amalgam in aqueous ethanol, since greater yields of the corresponding hydroxylaminoeveminomicin are produced thereby. Thus, for example, when utilizing everninomicin D as starting antibiotic, it is usually purified just prior to reduction with aluminum amalgam so as to obtain everninomicin D having the following physical constants: R, of 0.79 in a 60%-acetone-40% benzene system; Specific Optical Rotation: [111 34.2(chloroform); Neutralization Equivalent: I563 (Calculated neutralization equivalent for C H O NCl =l537) pKa: 7.2;

This is conveniently accomplished by reprecipitating everninomicin D at least twice in acetone/petroleum ether/ethyl ether followed by further purification of the reprecipitated everninomicin D by column chromatography using silica gel, eluting with acetone-benzene solvent mixtures, then combining the purest fractions as determined by thin layer chromatography. The purified everninomicin D is preferably stored underneath an atmosphere of nitrogen at about 5C to minimize undesirable side reactions such as hydrolysis and oxidation reactions.

We have found that a 50:50 alcoholzwater mixture is advantageously employed in our process, however, other alcoholzwater ratios may be used. In general, the amount of water in alcohol is limited by the solubility of the starting antibiotic, the reaction rate of our process being considerably slower when the starting everninomicin is insoluble in the solvent mixture. We heive found that everninomicin D remains essentially unchanged when treated via conventional reducing means, e.g., by hydrogenation in the presence of a catalyst such as palladium, Raney Nickel, palladium on charcoal, platinum, and the like. It is thus surprising that by our process an everninomicin containing a nitro group, e.g. everninomicin B, C, or D, is reduced by means of aluminum foil amalgam in aqueous alkanol to produce a product mixture comprising the corresponding nitrosoeveminomicin and hydroxylaminoeverninomicin.

Our reduction process is usually carried out under an inert atmosphere (e. g. under an atmosphere of argon or preferably nitrogen), although it may be carried out in the presence of air.

A convenient method of carrying out our reduction process comprises preparing a solution of purified everninomicin in an approximately 50:50 aqueous ethanol solvent mixture and adding aluminum amalgam freshly prepared from Reynolds Wrap aluminum foil, the amount of amalgam employed per gram of everninomicin starting compound usually being that prepared from up to two grams of aluminum (i.e. the weight of aluminum being up to twice that of starting antibiotic). The foregoing mixture is stirred at room temperature until all the amalgam disappears. The resulting product mixture comprising nitrosoeveminomicin and hydroxylaminoeverninomicin is then isolated by filtration and the products separated and purified utilizing known techniques such as recrystallization, reprecipitation, extraction, and chromatographic techniques. For example, a preferred species of our reduction process, i.e. that utilizing everninomicin D as starting antibiotic, is carried out by adding to a solution containing 4.5 grams purified everninomicin D in 100 ml. 95% ethanol and ml. water, freshly prepared aluminum amalgam derived from 810 mg. of Reynolds Wrap aluminum foil, and stirring the mixture at room temperature until the amalgam has disappeared (about two hours) and filtering off the resulting product mixture comprising nitrosoeveminomicin D and hydroxylaminoeveminomicin D. The hydroxylaminoeveminomicin D is conveniently isolated from the product mixture via thin layer chromatographic techniques such as that described in detail in Example 1D whereby the product mixture is chromatographed on silica gel preparative plates (2000 p. thick) using 50% acetone in benzene as developing solvent, followed by visualization of the plate under ultraviolet light, and removal of the band having an R;= 0.45 by means of acetone. Concentration of the acetone solution of the R;= 0.45 band yields purified hydroxylaminoeverninomicin D in about 33% yield having properties as set forth hereinbelow and in Example 1B.

When isolating nitrosoeveminomicin D from the product mixture comprising hydroxylaminoeverninomicin D and nitrosoeveminomicin D, the product mixture is usually chromatographed on silica gel preparative plates (2000 p. thick) using acetone/ethyl acetate/benzene (222:1) as developing solvent, visualizing the plate under ultraviolet light, then extracting with acetone that band having an R, value in the range of 0.75-0.85, followed by concentration of the acetone extracts to yield nitrosoeveminomicin D in small yields, i.e. less than 1%.

Oxidation Process:

As described hereinabove, low yields of nitrosoeverninomicin are obtained when an everninomicin selected from the group consisting of everninomicin B, everninomicin C, and everninomicin D is reduced with aluminum amalgam. Accordingly, the process of choice for preparing nitrosoeveminomicin is via oxidation of the corresponding hydroxylaminoeverninomicin utilizing reagents such as aerial oxidation in alkaline solution (e.g. bubbling air through a solution of hydroxylaminoeverninomicin D in dilute aqueous sodium hydroxide) or with an alkali metal hypobromite (preferably sodium hypobromite) in an aprotic solvent, the latter being the preferred oxidizing reagent for this process.

Alkali metal hypobromites which are useful in our oxidation process include potassium hypobromite, lithium hypobromite, and preferably, sodium hypobromite. Aprotic solvents useful in our oxidation process include dioxane, diethylene glycol dimethyl ether (also called diglyme), methanol, ethanol, and preferably, tetrahydrofuran.

Our oxidation process is usually carried out by treating one gram of hydroxylaminoeverninomicin (e.g. hydroxylaminoeveminomicin D) in 10 ml. tetrahydrofuran with an equal volume (i.e. 10 ml.) of aqueous sodium hypobromite prepared as described in Organic Synthesis, COLL, Vol. 4, 45 (1963) and stirring the reaction mixture until the starting hydroxylaminoeverninomicin has disappeared as determined via thin layer chromatographic techniques. The nitrosoeveminomicin thereby produced (e.g. nitrosoeveminomicin D) is isolated from the reaction mixture by addition of water followed by extraction with a halogenated solvent, e.g. methylene chloride, and evaporation of the methylene chloride extracts to a residue comprising about 60% yield of nitrosoeveminomicin. Further purification can be effected utilizing thin layer chromatographic techniques similar to that described hereinabove for the purification of nitrosoeveminomicin D, whereby is isolated about a 60% yield of nitrosoeveminomicin D having physical characteristics as set forth in Example 2A hereinbelow.

Composition-of-Matter Aspect Included within the composition-of-matter aspect of this invention are eveminomicin antibacterial derivatives selected from the group consisting of nitrosoeverninomicin B, nitrosoeveminomicin C, nitrosoeverninomicin D, hydroxylaminoeverninomicin B, hydroxylaminoeverninomicin C, hydroxylaminoeverninomi- :in D, nitrone derivatives of said hydroxylaminoeverninomicins B, C, and D, and pharmaceutically acceptable salts of the foregoing nitrosoeveminomicins and of the hydroxylaminoeveminomicins and their nitrones.

The above listed nitrosoeveminomicins, hydroxylaminoeverninomicins and nitrone derivatives of said aydroxylaminoeveminomicins all contain an acidic phenolic hydroxyl function which is readily convertible :o pharmaceutically acceptable cationic salts thereof itilizing procedures known in the art.

Among the pharmaceutically acceptable cationic aalts contemplated for this invention are salts of alkali and alkaline earth metals (e.g. sodium, potassium, :lacium, aluminum) and salts with an amine selected :rom the group consisting of trialkylamines, procaine, libenzylamine, N-benzyl-betaphenethylarnine, N,N'- libenzylethylene-diamine, N,N-bis-dehydroabietylthylenediamine and N-(lower)alkylpiperidines, e.g. I-ethylpiperidine. Also included within the term pharmaceutically acceptable salts are N-methyl glucamine salts of the nitrosoeveminomicins and of the lydroxylaminoeverninomicins and nitrone derivatives hereof.

The hydroxylaminoeverninomicin nitrone derivatives )f our invention are derived from the corresponding iydroxylaminoeverninomicin (e.g. hydroxylamino- :verninomicin B, C, or D) by treatment in known manner with an aldehyde which may have an aliphatic, ali- :yclic or aromatic hydrocarbon radicals which may be ubstituted by oxygen, sulfur or nitrogen in a side chain vr a hydrocarbon chain or ring forming hetero-aldelyde. Our nitrone derivatives are thus derived from alirhatic aldehydes such as formaldehyde, acetaldehyde, aropionaldehyde, heptanal, decanal, n-dodecanal, rom alicyclic aldehydes such as cyclopropylaldehyde, :yclopentylaldehyde, cyclohexylaldehyde, 3-cyclohex- 'lpropylaldehyde, from aromatic aldehydes such as enzaldehyde, p-hydroxybenzaldehyde, tolylaldehyde, C-phenethylaldehyde, naphthylaldehyde, a-naphthylformaldehyde, and from hetero substituted aldehydes such as furfural, thienylaldehyde, thiazolaldehyde, pyrazinaldehyde, pyranylaldehyde, and the like. Our nitrone derivatives are preferably derived from aldehydes having hydrocarbon radicals or oxygen containing radicals including benzaldehyde, heptylaldehyde, and furfural.

The eveminomicin B, C, and D reduction products of this invention are oligosaccharides, and each contains a dichloroisoeverninoyl ester grouping, a reduced evernitrose group (i.e. a group wherein the nitro function in evernitrose has been reduced to nitroso or hydroxylamino) or derivatives thereof, at least two ortho ester functions, and several anomerically linked monosaccharide groups.

It is known in the art that ortho esters hydrolyze readily and that hydroxylamino functions oxidize easily to a nitroso function. In view of the presence of these functions in our compounds, to minimize side reactions, it is preferable to store them under anhydrous conditions in an inert atmosphere (e.g. under nitrogen, argon, etc.) at cool temperatures (i.e. at about 5l0C).

Preferred compounds of our invention include those derived from eveminomicin D and derivatives thereof, all of which possess antibacterial activity, being particularly effective in inhibiting the growth of gram-positive bacteria. Of these, hydroxylaminoeverninomicin D and the pharmaceutically acceptable salts thereof, particularly the sodium salt, are of special interest since they possess comparable antibacterial activity against gram-positive bacteria as that exhibited by the everninomicin D precursor; moreover, when administered by injection in the form of its sodium salt, hydroxylaminoeverninomicin D is advantageously more rapidly absorbed, producing higher serum levels, and more rapidly excreted than eveminomicin D. Advantageously, certain gram-negative bacteria are also susceptible to (i.e. their growth is inhibited by) hydroxylaminoeverninomicin D.

Included among the preferred eveminomicin D reduction products of our invention are compounds defined by structural formula 1 shown hereinabove and the pharmaceutically acceptable salts thereof wherein Y is hydrogen, Z is and X is a member selected from the group consisting of nitroso (i.e. X is N=0), hydroxylamino (i.e. X is and a hydroxylamino nitrone derivative (i.e. wherein X is wherein R is a hydrocarbon which may be substituted by oxygen, sulfur, or nitrogen.

The compound of formula I wherein Y is hydrogen, Z 18 ocii and X is -N= is nitrosoeverninomicin D; the compound of formula I wherein Y is hydrogen, Z is andXis is hydroxylaminoeverninomicin D, and those comare hydroxylaminoeverninomicin D nitrone derivatives including hydroxylaminoeveminomicin D benzyl nitrone (derivative wherein R is phenyl); hydroxylaminoeverninomicin D furfuryl nitrone (derivative wherein R is furfuryl); and hydroxylaminoeverninomicin D heptyl nitrone (derivative wherein R is hexyl).

The pharmaceutically acceptable salts of the compounds of our invention include those which replace the phenolic hydrogen with a cation (e.g. hydroxylaminoeverninomicin D sodium salt) and those wherein the phenolic hydrogen and everninomicin radical forms an acid addition salt with an amine (e.g. nitrosoeverninomicin D N-methylglucamine salt).

Other pharmaceutically acceptable salts of our invention are:

hydroxylaminoeveminomicin D sodium salt;

hydroxylaminoeverninomicin D N-methylglucamine salt;

hydroxy]aminoeverninomicin D benzyl nitrone sodium salt;

hydroxylaminoeverninomicin D benzyl nitrone N- methylglucamine salt; hydroxylaminoeverninomicin D heptyl nitrone sodium salt;

hydroxylaminoeverninomicin D heptyl nitrone N- methylglucamine salt; hydroxylaminoeveminomicin D furfuryl nitrone sodium salt; and

hydroxylaminoeverninomicin D furfuryl nitrone N- methylglucamine salt.

The pharmaceutically acceptable cationic salts (e.g. the sodium salt) are prepared according to known procedures such as by combining equimolar quantities of the corresponding base (e.g. sodium hydroxide) to the everninomicin derivative (e.g. hydroxylaminoeverninomicin D) in an aqueous solution and lyophilizing the resultant solution of the hydroxylaminoeverninomicin D salt.

The pharmaceutically acceptable salts of our invention are white amorphous solids which are water soluble and fonn stable aqueous solutions. They are, therefore, a particularly preferred species of our invention since they are a convenient vehicle for administering the nitrosoeverninomicins and hydroxylaminoeverninomicins of our invention. A particularly valuable species of our invention is hydroxylaminoeverninomicin D sodium salt.

The nitrosoeverninomicins B, C and D and hydroxylaminoeverninomicins B,C and D of this invention including derivatives and pharmaceutically acceptable salts thereof, particularly the preferred group of compounds of our invention, i.e. nitrosoeverninomicin D, hydroxylaminoeverninomicin D, and nitrones thereof, and pharmaceutically acceptable salts of the foregoing, exhibit a narrow spectrum antibacterial activity in vitro against grampositive bacteria (e.g. Staphylococcus aureus, Staph. 11631, Staph. W., Streptococcus pyogenes C, Strep. pyogenes C-203 and Bacillus subtilis) and against certain gram-negative bacteria (e.g. Neisseria gonorrhea and Neisseria meningitidis). Our compounds are thus advantageously employed as laboratory reagents when attempting to determine the presence of enteric gram-negative organisms. They may be used to inhibit overgrowth of such organisms in culture media, either alone or in combination with other antibacterial agents to reduce or eliminate the heavy overgrowth of gram-positive organisms permitting the determination of gram-negative organisms such as Klebsiella pneum0- niae or Escherichia coli in cultures obtained in diagnostic procedures. As such reagents they may be employed in solution.

In view of their action against gram-positive and gram-negative organisms, the antibacterials described herein may be used to sterilize equipment such as in operating rooms and in hospital wards.

The comparative in vitro activities of nitrosoeverninomicin D and the sodium salt thereof, hydroxylaminoeverninomicin D and the sodium sit and N- methylglucamine salt thereof, hydroxylaminoeverninomicin D benzyl nitrone N-methylglucamine salt, hydroxylaminoeverninomicin B and hydroxylaminoeverninomicin C are set forth in Table 1 below.

The susceptibility of the test gram-positive microorganisms to the antibacterials was determined by standard tube dilution methods. In each instance 10" dilutions of 24-hour broth cultures were employed as inoculum with the endpoints being taken after incubation for 24 hours at 37C in a Difco Penassy broth medium (Difco Labs, Detroit, Mich.). In the tables, the activity (or assay value) of each respective antibacterial agent is expressed in units per milliliter or micrograms per milliliter.

The microbiological assay (also called the bioassay) is effected microbiologically by a standard cup-type agar diffusion assay technique using S. aureus (A.T.C.C. 6538p) as test organism. A reference curve is prepared by plotting the dosage-response of the antibacterial agent in ethanol diluted by phosphate buffer at pl-l7 upon a medium of the following composition which has been inoculated with the test organism (i.e. S. aureus):

Percent Peptone 0.6 Pancreatic digest or casein 0.4 Yeast extract 0.3 Beef extract 0. l5

11 12 cominued It is apparent from the foregoing data that, when ad- Dextrose 0.15 ministered by injection in the form of its sodium salt, Agar hydroxylaminoeverninomicin D exhibits antibacterial activity comparable to that of the everninomicin D pre- 5 cursor and, advantageously, is more rapidly absorbed A suspension of the assay organism (S. aureus producing higher serum levels (i.e. 20-30) in the dog A.T.C.C. 6538p) is standardized to produce 20% transthan that produced by everninomicin D (i.e. 2-3). mission at 660 my. in a colorimeter. The potency of the We have described in detail the preferred species of Sample is determined from the refefencefiufve and our invention relating to hydroxylaminoeverninomicin Pressed in terms of units P milligram unit being 10 D derivatives. In similar manner, our invention is carthat amount of test substance required to produce a ried out to produce antibacterial substances which are mm zone of inhibition with a steel cylinder of 6.5 mm derivatives of everninomicin B and everninomicin C. outside diameter). Thus, an everninomicin antibiotic mixture produced The susceptibility of the gram-negative microorganwhen Micromonospora carbonacea var. carbonacea or isms to the antibacterials was determined by plate dilul5 v i th f i subjected to submerged aerobic fertion methods in Th yer-M rtin me ium by heknown mentation according to known procedures. Said ever- Thayer-Martin procedure. ninomicin mixture, comprising everninomicin B, ever- TABLE 1 EVERNINOMICIN DERIVATIVES Evemino- Hydroxyl- Sodium salt Nitrose- Sodium salt N-methylgluca- Hydroxyl- Hydroxylmicin D aminoeverand N-methyleveminoof nitrosomine salt of aminoeveraminoeverninomicin glucamine salt micin eveminomihydroxylaminoninomicin ninomicin of hydroxyl- D cin D everninomicin C B aminoevemino- D benzyl micin D nitrone MIC (meg/ml) 0.030.3 0.3-0.8 0.08O.8 0.080.8 0.08-3 0.ll O.l0.8 MlC (meg/ml) 0.2-1.0 PD mg/kg Oral 15 25 SC. 5 0.5-5 LD in mice LP. 3800 500 (mg/kg) s.c. 3800 500 Oral 3800 2000 Lv. 125 30 Peak serum levels in dogs (meg/ml) 10 mg/kg I.M. 2-3 -30 6 1-2 15 Peak serum levels in rats (meg/ml) mg/kg s.c. 10-12 -40 10-11 10-12 Staphylococcus and Slrepmcoccus "N. gulmn'lma and N. meningitidis In above Table 1, there is also listed in vivo data for the peak serum levels in dogs and rats, as well as PD and LD doses in rats for everninomicin D and reduced derivatives thereof, against Staphylococcus and Streptococcus.

The acute toxicity (LD of the antibacterial substances in either aqueous suspensions or aqueous solutions is measured in milligrams per kilogram in standard manner using male CF-l mice weighing 20 grams.

The protection tests to determine the dose required to protect fifty percent of the animals tested (PD use groups of seven mice (male albino CF-l mice, each weighing 18-20 g.) each at five dose levels with 10 mice serving as controls. Mice were each treated with a single subcutaneous or oral dose 1 hour after intraperitoneal infection with approximately 10 organisms. Control mice were generally dead 18-24 hours after infection. Survivors in treated groups were determined 48 hours after infection. Probit procedures were used to determine PD values in milligrams per kilogram.

In determining peak serum leves, the animal tested (e.g. dog or rat) is given a single dose of 10 or 25 milligrams of everninomicin D or derivative thereof of this invention, blood samples are taken periodically, and the serum thereof is assayed for antibacterial activity by an agar-diflusion-assay as described by Weinstein et al., Antimicrobial Agents and Chemotherapy, p 24 (1964).

ninomicin C and everninomicin D, is purified and the B, C, and D components are separated utilizing chromatographic techniques such as described hereinbelow in Example 8. The purified everninomicin B and everninomicin C, respectively, having the physical constants set forth in Examples 8C(3) and 8C(2), respectively, are then reduced with aluminum amalgam in an alcohol-aqueous mixture according to our process to produce hydroxylaminoeveminomicin C, respectively, having antibacterial activity as evidenced by data set forth in Table I determined by standard in vitro and in vivo tests discussed hereinabove. Upon oxidizing the hydroxylaminoeveminomicin B and C derivatives thereby produced with sodium hypobromite in tetrahydrofuran by the oxidation process of this invention, there is obtained nitrosoeverninomicin B and nitrosoeverninomicin C, respectively. Nitrone derivatives of hydroxylaminoeveminomicin B and C having antibacterial activity are derived as described herein by treatment of the respective hydroxylaminoeverninomicin with an aldehyde. Salts of hydroxylaminoeveminomicin B and C including cationic salts, amine salts, and N-methylglucamine salts, are prepared in manner similar to that described for everninomicin D.

As discussed hereinabove, the everninomicin B, C, and D reduction-products of this invention exhibit anti' bacterial activity against gram-positive bacteria and against certain gram-negative bacteria. Advantageously, our everninomicin B, C, and D reduction 13 products exhibit antibacterial activity against bacteria which are resistant to other antibacterial agents such as penicillin, penicillinase-resistant penicillins, tetracycline. macrolide antibacterials such as erythromycin, and lincomycin.

Procedures are set forth hereinbelow to illustrate the best mode contemplated by applicants for carrying out their invention and are not to be construed as limiting the scope thereof.

EXAMPLE 1 Reduction of Everninomicin D to Hydroxylaminoeverninomicin D A. Purification of Everninomicin D 1. Everninomicin D, the requisite intermediate, is prepared by cultivating a microorganism selected from the group consisting of Micromonospora carbonacea var. carbonacea NRRL 2972 and Micromonospora carbonacea var. (ll rantiaca NRRL 2992 under submerged aerobic conditions in an aqueous nutrient medium containing an assimilable source of carbon and nitrogen and isolating everninomicin D from the antibiotic mixture thereby produced by procedures described in U.S. Pat. No. 3,499,078.

2. Treat a solution of 66 g. of everninomicin D (prepared as described in paragraph 1 hereinabove) in 300 ml. of acetone with 7 g. of charcoal at room temperature. Filter and wash the charcoal with 100 ml. of acetone. Pour the combined acetone filtrate and washes onto a solution of 10.5 liters of petroleum ether (b.p. 60C) and 2.7 liters of ethyl ether. Filter and dry the resultant precipitate of everninomicin D.

3. Further purify the thus obtained everninomicin D (about 56 grams) by reprecipitating an acetone (250 ml.) solution thereof with about liters of a solvent mixture comprising petroleum ether/ethyl ether (4:2). Filter and dry the resultant precipitate of everninomicin D to a constant weight at 60C (yield about 52 grams).

4. Further purify 30 g. of the everninomicin D obtained as described above in a minimum amount of acetone, add 120 g. of fresh silica gel, then evaporate the solvent at 60C until a constant weight is obtained. Add this mixture to the top of a 5 ft. by 1 inch chromatographic column charged with 450 g. of silica gel made up in benzene. Elute the column first with about 7 liters of a solvent mixture comprising acetone-85% benzene, followed by elution with a solvent mixture comprising acetone-80% benzene. Collect 1 liter fractions, monitoring the fractions by thin layer chromatography. Concentrate pooled fractions 8-16 in vacuo at 50C to a residue comprising everninomicin D (about 18 g.) having an R, of 0.79 via thin layer chromatography in a 60% acetone-40% benzene system. Molecular Specific Optical Rotation: [01],, 34.2 (chloroform) 14 Neutralization Equivalent: C l-l O NCl 15 37).

pKa: 7.2 5. Store everninomicin D under an atmosphere of nitrogen at about 5C.

1563 D (calc. for

B. Preparation of Aluminum Amalgam C. Reduction of Eveminomicin D To a solution of 4.5 g. of everninomicin D (purified as described in Example 1A) in 100 m1. of 95% ethanol and ml. of water, add with stirring at room temperature aluminum amalgam prepared from 810 mg. of Reynolds Wrap Aluminum Foil as described in Example 1B. Stir the reaction mixture at room temperature until all the amalgam disappears (approximately 2 hours). Filter the gray reaction mixture on a Buchner funnel using a filter pad and wash the insoluble residue with ethanol. combine the filtrate and ethanol washings and concentrate to a volume of approximately ml. Cool the concentrate in an ice bath for about an hour; separate by filtration the resulting insoluble fraction comprising 2.9 g. of hydroxylaminoeverninomicin D; dry the insoluble fraction in vacuo at room temperature prior to further purification.

D. Purification of Hydroxylaminoeverninomicin D Combustion Analysis Found Cale.

Carbon 51.81% 52.05 Hydrogen 6.64% 6.68 Nitrogen 0.59% 0.92 Chlorine 4.55% 4.66

Specific optical rotation: [01],, 39.3 (chloroform);

A methanol max Ultraviolet absorption: 215 m;:., e,', [9.2 3 4 infrared absorption spectrum in chloroform: 2.92, 5.80, 6.37, 6.92. 7.13, 7.26, 8.06, (broad), broad absorption between 9.13 and 9.70 microns (see FIG. 1).

In this specification, when reporting ultraviolet absorption data, by e, is meant e which is defined as the optical density of a 1% solution of a compound measured in a l centimeter cell.

Nuclear magnetic resonance spectrum in deuterochloroform: (See FIG. 2).

EXAMPLE 2 Nitrosoeve rninomicin D A. Preparation by Oxidation of l-Iydroxylaminoeverninomicin D The reagent sodium hypobromite is prepared according to the procedure described in Organic Synthesis, COLL., Vol. 4, 45 (1963).

To 1 millimole (1 mmol) of a solution of hydroxylaminoeverninomicin D in solution in tetrahydrofuran (l g. dissolved in 10 ml. of tetrahydrofuran), add 1 mmol of aqueous sodium hypobromite solution at room temperature. Follow the course of the reaction by thin layer chromatography (silica gel plates 250 n; solvent system acetone/ethyl acetate/benzene [2:2111). After the starting material has disappeared, add water to the reaction mixture and extract with methylene chloride. Evaporate the combined methylene chloride. Evaporate the combined methylene chloride extracts to a residue comprising nitrosoeverninomicin D (yield about 60% theory) which is further purified as described hereinbelow.

Chromatograph 100 mg. of nitrosoeverninomicin D on a thin layer chromatographic preparative plate (2000 ,u. thick) using acetone/ethyl acetate/benzene (2:2:1 as the developing solvent. Visualize the purified nitrosoeverninomicin D by means of ultraviolet light and. extract from the plate with acetone the band in the approximate R range of 0.75-0.85. Evaporate the combined acetone extracts to a residue comprising about 60 mg. of nitrosoeverninomicin D having the following properties:

Molecular Formula: C I-I O NCI Molecular Weight: 1521 (Calcd). m.p.: 163-165C Kofler block Combustion Analysis: Found Calc.

Carbon 51.66% 52.11 Hydrogen 6.52% 6.56 Nitrogen 0.84% 0.92 Chlorine 4.50% 4.66

Specific Optical Rotation: [(11 30.8 (chloroform);

A methanol max Ultraviolet Absorption 212 mu, s 16.05 Spectrum:

292 my, E, 5.60

Infrared Absorption Spectrum in chloroform: 2.9, 3.4, 5.8, 6.4, 6.5, 6.9, 7.2, 9.0, 9.6, 10.2, 10.5 microns (See FIG. 3). Nuclear magnetic Rsonance Spectrum: (See FIG. 4).

Bioassay: 1460 'y/ml.

B. Preparation by Reduction of Everninomicin D Isolate the purify the nitrosoeverninomicin D using 20 silica gel preparative plates (2000 thick) and using acetone/ethyl acetate/benzene (2:2: 1) as the developing solvent. Visualize the purified nitrosoeverninomicin D (R, about 0.78) using an ultraviolet light. Extract the 0.78 R; band from the plate using acetone. Combine the acetone extracts and distill in vacuo to a residue of nitrosoeverninomicin D.

EXAMPLE 3 Hydroxylaminoeverninomicin D Benzyl Nitrone To 400 mg. of hydroxylaminoeverninomicin D in 15 ml. of anhydrous ethanol, add 0.2 ml. of benzaldehyde. Stir at room temperature for 72 hours, then distill in vacuo to a residue. Triturate the residue with hexane and separate by filtration and air dry the resultant precipitate comprising hydroxylaminoeverninomicin D benzyl nitrone.

Purify via thin layer chromatographic techniques using silica gel with acetone/benzene (1:1) as the developing solvent. Visualize the benzyl nitrone derivative using ultraviolet light and extract with acetone the band within the R, zone of from about 0.5 to about 0.7. Evaporate the acetone in vacuo to a residue of 200 mg. of purified hydroxylaminoeverninomicin D benzyl nitrone as a colorless solid having the following characterizing data:

m.p.: l68l70C Kofler block;

Specific Optical Rotation: a 26 67 (chloroform);

Ultraviolet Absorption Spectrum: D A 295 m .L, A ,1495;

Infrared Absorption Spectrum in chloroform: 2.9, 3.4, 5.75, 5.8, 6.35, 6.9, 7.2, 9.1, 9.6, 10.5, 11.0 microns (See FIG. 5).

Bioassay: 1309.0 7 /ml.

EXAMPLE 4 I-Iydroxylaminoeverninomicin D Furfuryl Nitrone To 375 mg. of hydroxylaminoeverninomicin D in 25 ml. of anhydrous ethanol, add 1 ml. of Z-furancarbonal (2-furfuraldehyde). Stir at room temperature overnight then distill in vacuo to a residue. Triturate the residue with hexane and dry the resultant precipitate comprising hydroxylaminoeverninomicin D furfuryl nitrone.

Purify via preparative thin layer chromatography utilizing silica gel with acetone/benzene (1:1) as the developing solvent. Visualize the furfuryl nitrone derivative by means of ultraviolet light and extract with acetone that band having an R, in the range of from about 0.75 to about 0.85. Concentrate the acetone solution in vacuo to obtain 186 mg. (50% yield) of hydroxylaminoeverninomicin D furfuryl nitrone having the following characterizing data:

Molecular Formula: C I-I NO Cl 2H O; Molecular Weight: 1637 (Calc.)

Combustion Analysis: Found Calc.

Carbon l .8 1% 52.09 Hydrogen 6.70% 6.59 Nitrogen 0.74% 0.86 Chlorine 4.04% 4.33

Specific Optical Rotation: a 25 63.2 (chloroform);

Ultraviolet Absorption Spectrum: A 302 mu, 6,, 18.35;

Infrared Absorption Spectrum: 2.9, 3.4, 5.8, 6.2, 6.3, 6.8, 7.2, 8.0, 9.0, 9.6, 10.5, 10.9 microns (See FIG. 6).

Bioassay: 1063.4 y /ml.

EXAMPLE 5 Hydroxylaminoeverninomicin D Heptyl Nitrone Molecular Formula: c,,r-r,,,No,,c1,; Molecular Weight: 1619 (c2116.)

Combustion Analysis: Found Calc.

Carbon 54.15% 54.12 Hydrogen 7.44% 7.03 Nitrogen 0.72% 0.86 Chlon'ne 3.84% 4.38

Specific Optical Rotation: a 26 46.0 (chloroform);

Ultraviolet Absorption Spectrum: A 295 mp4,, 5.25;

Infrared Absorption Spectrum in Chloroform: 2.9, 3.5, 5.7, 6.3, 6.9, 7.2, 9.0, 9.6, 10.5, 10.9 microns (See FIG. 7).

Bioassay: 892 7 /ml.

EXAMPLE 6 Sodium Salt of Hydroxylaminoeveminomicin D and Derivatives Thereof A. To a vigorously stirred suspension of l g. of hydroxylaminoeveminomicin D in 25 ml. of water under a nitrogen atmosphere, slowly add 0.1 N-sodium hydroxide (about 6.8 ml.) until the pH of the reaction mixture is 9.5 and the solid is in solution. Stir at room temperature for an additional hour (final pH about 8.5). Lyophilize the clear solution to obtain 0.95 g. of hydroxylaminoeverninomicin D sodium salt as a white solid.

B. In the above procedure by substituting for the sodium hydroxide equimolar quantities of other alkaline metal hydroxides (e.g. potassium hydroxide and lithium hydroxide) or by alkaline earth metal hydroxides (e.g. calcium hydroxide and barium hydroxide), there is obtained the corresponding alkali metal or alkaline earth metal salt, e.g. hydroxylaminoeveminomicin D potassium salt, hydroxylaminoeveminomicin D lithium salt, hydroxylaminoeverninomicin D calcium salt and 18 hydroxylaminoeverninomicin D barium salt, respec tively.

C. In a manner similar to that described in Example 6A hereinabove, treat each of the following compounds with 0.1 N-sodium hydroxide:

nitrosoeverninomicin D;

hydroxylaminoeverninomicin D benzyl nitrone;

hydroxylaminoeverninomicin D furfuryl nitrone; and

EXAMPLE 7 N-Methyl Glucamine Salt of Hydroxylaminoeverninomicin D and Derivatives Thereof A. To 300 mg. of hydroxylaminoeverninomicin D dissolved in 1.5 ml. of methanol, add 40 mg. of N- methyl glucamine and stir the reaction mixture at room temperature for 1 /2 hours. Slowly add ether (40 ml.) with good agitation. Separate the resultant precipitate by filtration and air dry to give mg. of hydroxylaminoeverninomicin D N-methyl glucamine as a white solid.

Similarly, treat each of the following with N-methyl glucamine in methanol:

nitrosoeverninomicin D;

hydroxylaminoeverninomicin D benzyl nitrone;

hydroxylaminoeverninomicin D furfuryl nitrone; and hydroxylaminoeverninomicin D heptyl nitrone.

Isolate the resultant product in a manner similar to that described hereinabove to obtain, respectively,

nitrosoeverninomicin D N-methyl glucamine salt;

hydroxylaminoeverninomicin D benzyl nitrone N- methyl glucamine salt;

hydroxylaminoeveminomicin D furfuryl nitrone N- methyl glucamine salt; and

hydroxylaminoeverninomicin D heptyl nitrone N- methyl glucamine salt.

EXAMPLE 8 Preparation and Purification of Everninomicin B, Everninomicin C, and Everninomicin D A. Preparation of Antibiotic Mixture Comprising Everninomicin B, Everninomicin C, and Everninomicin D In the manner described in US. Pat. No. 3,499,078, Example 1, prepare a 500 gallon fermentation broth by cultivating a micro-organism selected from the group consisting of Micromonospora carbonacea var. carbonacea NRRL 2972 and Micromonospora carbonacea var. Aurantiaca NRRL 2992 under submerged aerobic conditions in an aqueous nutrient medium containing an assimilable source of carbon and nitrogen. Extract 500 gallons of fermentation broth with 500 gallons of methylene chloride, then concentrate the methylene chloride extracts in vacuo to a residue having a volume of about 3 liters. Pour the residue into 30 liters of hexane, then filter and air dry the resultant precipitate comprising a mixture of everninomicin B, everninomicin C, and everninomicin D.

B. Separation of Antibiotic Mixture Prepare the chromatographic column by making a slurry of 11.35 kg. of silica gel (Bakers) in benzenelacetone (60:40). Then transfer the slurry into two ft. X 4 in. columns and allow the columns to settle over night. Place the two columns in series so they will act as one continuous column.

Dissolve 300 g. of the mixture of everninomicin B, C and D prepared as described in Example 8A in about 1 liter of benzene/acetone (60:40) with vigorous stirring. Transfer this solution onto the columns.

Elute the column (i.e. the two columns in series) with benzene/acetone (60:40) and collect 1800 ml. fractions. Combine like fractions as determined by thin layer chromatographic analysis (silica gel: benzene: acetone (60:40)). Then evaporate the combined fractions to a residue comprising everninomicin D, everninomicin C and everninomicin B, respectively, as follows:

Fractions 14-18 yield a residue comprising 76.9 g. of everninomicin D; fraction 21 yields a residue comprising 5 g. everninomicin C and combined fractions 22-27 yield a residue comprising 29.5 g. of everninomicin B.

C. Purification of Everninomicin D,C, and B,

respectively 1. Everninomicin D Purify the residue of fractions 14-18 obtained in above Example 88 in a manner similar to that described in Example 1A to obtain purified everninomicin D having the physical constants set forth in Example 1A(4).

2. Everninomicin C Combustion Analysis: Found Calc'd Carbon 52.07 51.56% Hydrogen 6.44 6.49 Nitrogen 0.51 0.91 Chlorine 4.72 4.61

Specific ()ptical Rotation: a 33.7 (chloroform);

A methanol max Ultraviolet Absorption: 208 my, 6 l

Infrared Absorption Spectrum in chloroform: 2.9, 3.4 (broad), 5.75, 6.32, 6.45, 6.85, 7.1, 7.2, 7.4, 7.7, 8.0, 9.0, 9.6, 10.2, and 11.0 microns (See FIG. 8).

3. Everninomicin B Chromatograph 28 g. of everninomicin B prepared as described in above Example 88 (i.e. the residue from fractions 22 to 27) on 900 g. of silica gel G (TLC Grade; according to Stahl) eluting with acetone in benzene. Collect 20 ml. fractions and combine like 20 fractions as determined by thin layer chromatographic analysis. Evaporate combined fractions 211 to 310 in vacuo to a residue comprising purified everninomicin B (12.6 g.) having the following properties:

Specific Optical Rotation: a L, -33(chloroform) 210 mu, 6,, 17 285 mu, e 1.5

A methanol max Ultraviolet Absorption Spectrum:

Infrared Absorption Spectrum (in chloroform): 2.9, 3.4, 5.75, 6.3, 6.45, 6.85, 7.20, 8.0, 8.5, 9.0, 9.6, 10.2, and 10.5 microns.

EXAMPLE 9 Reduction of Everninomicin C to Hydroxylaminoeverninomicin C A. In a manner similar to that described in Example 1C, treat everninomicin C (prepared and purified as described in Example 8) in aqueous ethanol with aluminum amalgam. Isolate the resultant product in a manner similar to that described to obtain hydroxylaminoeverninomicin C.

B. Purify the crude hydroxylaminoeveminomicin C obtained as described in above ExampleQA in a manner similar to that described in Example 1D using silica gel preparative plates (2000 u thick) and using 50% acetone-benzene as the developing solvent. Visualize the purified hydroxylaminoeveminomicin C (R, 0.27) with ultraviolet light and extract the purified hydroxylaminoeverninomicin C band from the plate using acetone. Combine the acetone extract and distill to a residue comprising purified hydroxylaminoeverninomicin C having the following properties:

Molecular Formula: C H O NCl m.p.: -l68C Kofler block:

Combustion Analysis Found Calc'd Carbon- 51.35, 51.74% 51.64% Hydrogen 2 6.66, 6.75% 6.54 Nitrogen 0.93, 1.09% 0.96 Chlorine 5.75% 4.84

21 D. In a manner similar to that described in Example 7, treat hydroxylaminoeverninomicin C in methanol with N-methylglucamine to obtain hydroxylaminoeverninomicin C N-methylglucamine.

EXAMPLE Reduction of Eveminomicin B to Hydroxylaminoeverninomicin B A. In a manner similar to that described in Example 1C treat everninomicin B (prepared and purified as described in Example 8) in aqueous ethanol with aluminum amalgam. Isolate and purify the resultant product in a manner similar to that described in Example 1C to obtain a product comprising hydroxylaminoeverninomicin B.

B. In a manner similar to that described in Example ID, purify the crude hydroxylaminoeveminomicin B prepared in above Example 10A using silica gel preparative plates (2000 p. thick) and using 50% acetone in benzene as the developing solvent. Visualize the purified hydroxylaminoeverninomicin B (R, 0.18) using ultraviolet light and extract the purified hydroxylaminoeverninomicin B band from the plate using acetone. Combine the acetone extracts and distill to a residue comprising purified hydroxylaminoeverninomicin B having the following properties:

Molecular Formula: C H O NCl Molecular Weight: 1535 (Calc.)

Combustion Found: Calc. (for C I-I O NCl AI-I O) Analysis:

Carbon 49.61% 50.3

Hydrogen 6.63% 6.97

Nitrogen 1.00% 0.89

Chlorine Specific Optical Rotation: a -19.9 (methanol) A methanol 215 mpc 17.15 299 my 5,, 3.15

Ultraviolet Absorption Spectrum:

Infrared Absorption Spectrum in chloroform: 2.9,

3.4, 5.8, 6.35, 6.9, 7.2, 8.0, 9.1 (broad S), 9.6 (broad-S), 10.5, 11.0 microns (See FIG. 10).

C. In a manner similar to that described in Example 6, treat hydroxyleveminomicin B with sodium hydroxide to obtain hydroxylaminoeverninomicin B sodium salt.

EXAMPLE ll Nitrosoeverninomicins B and C EXAMPLE 12 Nitrone Derivatives of Hydroxylaminoeverninomicins B and C In a manner similar to that described in Examples 3 to 5 treat each of hydroxylaminoeverninomicin B and hydroxylaminoeveminomicin C in anhydrous ethanol with benzaldehyde, 2-furfuraldehyde and heptaldehyde, respectively.

Isolate and purify each of the resultant products in a manner similar to that described in Examples 3-5 to obtain the benzyl nitrone, furfuryl nitrone and the heptyl nitrone derivatives, respectively, of hydroxylaminoeverninomicin B and hydroxylaminoeveminomicin C, respectively.

The present invention includes within its scope pharmaceutical compositions comprising our novel reduction products of eveminomicins B, C, and D in association with a compatible, pharmaceutically acceptable carrier or coating. Also included within our invention is the method of eliciting an antibacterial response in a warm-blooded animal having a susceptible bacterial infection which comprises administering to said animal a non-toxic, antibacterially effective amount of a member selected from the group consisting of nitrosoeverninomicin B, nitrosoeverninomicin C, nitrosoeverninomicin D, hydroxylaminoeveminomicin B, hydroxylaminoeverninomicin D, hydroxylaminoeveminomicin D, nitrone derivatives of said hydroxyaminoeverninomicins, and pharmaceutically acceptable salts of the foregoing.

The compositions may be administered parenterally, orally, or topically, parenteral administration being preferred. In carrying out the methods of this invention, the active ingredient is normally combined with conventional pharmaceutical diluents and carriers which are based upon the desired route of adminstration. The parenteral route is preferred, particularly with salts of hydroxylaminoeverninomicin D, due to the increased absorption and rapid excretion when adminstered parenterally of our everninomicin reduction products as compared with their everninomicin B,C, and D precursors. In carrying out the method, the active group can, if desired, be combined with other therapeutically active compositions customarily included in antibacterial formulations.

The individual unit dosage and frequency of administration is determined not only by the nature and severity of the bacterial infection for which relief is sought, but also upon age, weight, species, underlying physical condition and route of administration. The exact amount to be administered should be nontoxic, yet pharmaceutically effective in alleviatig the symptoms of bacterial infections. Generally, for the treatment of bacterial infections, the compositions are administered parenterally so as to give a daily dose of from 1 to about 15 mgm/kg of a reduced everninomicin compound of this invention.

The following example illustrates a pharmaceutical composition according to the invention.

EXAMPLE l3 Parenteral Solution Mg. Hydroxylaminoeveminomicin D sodium salt 58.14 Sodium chloride 5.0 Sodium bisulfite 1.625 Sodium hydroxide (IN to pH 8.7) 0.56

Parenteral Solution-continued Purified Water, q.s. 1 ml.

Procedure Sparge with nitrogen about 90% of the required amount of water. With stirring, add the sodium chloide and sodium bisulfite and stir until dissolved. Add

he hydroxylaminoeveminomicin D sodium salt and vith stirring add 1N sodium hydroxide until the soluion reaches a pH of 8.7. Add water to make a total volime of 1 ml. filter through a sterilizing membrane.

With this solution, fill multiple dose vials. Lyophilize.

Sparge with nitrogen about 90% of the required mount of water and heat the water to a temperature of lbOllt 70C. Add the methylparaben and propylaraben, stir until dissolved, then cool the solution to !530C. Then follow the procedure described in Ex- 35 .mple l3.

EXAMPLE 15 Parenteral Suspension 0 Parenteral Suspension: Mg.

Hydroxylaminoeveminomicin D 100 Sodium carboxymethylcellulose 3 Polysorbate USP 1 Methylparaben 3.6 45 Propylparaben 0.4 Water, q.s. 2.0 ml.

OCH c1 3 5? HO C\ CH cl CH 3 o 3 c CH X Procedure 2. Aseptically, slurry hydroxylaminoeverninomicin 7 24 with a portion of the above vehicle and pass through a colloid mill.

3. Mix the milled slurry with the remainder of the vehicle.

4.Fill into sterile vials.

We claim:

1. The process which comprises treating an everninomicin antibiotic selected from the group consisting of eveminomicin B, eveminomicin C, and eveminomicin D with aluminum amalgam in aqueous lower alkanol at room temperature whereby is produced a product comprising a member selected from the group consisting of the corresponding hydroxylaminoeverninomicin, the corresponding nitrosoeveminomicin, and mixtures thereof.

2. The process according to claim 1 including the added step of isolating the hydroxylaminoeveminomicin derivative thereby produced.

3. The process according to claim 1 wherein said aluminum amalgam is an amalgam of Reynolds Wrap aluminum foil.

4. The process according to claim 1 wherein said aluminum amalgam is an amalgam of Reynolds Wrap aluminum foil and wherein said aqueous lower alkanol is aqueous ethanol.

5. The process for the preparation of nitrosoeverninomicin selected from the group consisting of nitrosoeveminomicin B, nitrosoeveminomicin C, and nitrosoeveminomicin D which comprises treating the corresponding hydroxylaminoeveminomicin selected from the group consisting of hydroxylaminoeverninomicin b, hydroxylaminoeveminomicin C, and bydroxylaminoeverninomicin D with an oxidizing reagent selected from the group consisting of aerial oxidation in an alkaline solution, and an alkali metal hypobromite in an aprotic solvent.

6. The process according to claim 5 wherein said oxidizing reagent is sodium hypobromite in tetrahydrofuran.

7. A compound selected from the group consisting of nitrosoeveminomicin B, nitrosoeveminomicin C, nitrosoeveminomicin D, hydroxylaminoeveminomicin B, hydroxylaminoeveminomicin C, hydroxylaminoeverninomicin D, nitrone derivatives of said hydroxylaminoeveminomicins B, C, and D, said compound having the following structural formula:

cu ochl3 z 3 i on 3 O c3 0 3 CH2 0 Q o o o 0 V 0H OCH ca; on 3 wherein X is a member selected from the group consisting of and wherein R is a hydrocarbon having up to 12 carbon atoms; Y is hydrogen or hydroxy; and

on 1 is CH/ 3 ocn,

or, provided Y is hydrogen, Z is hydrogen; and the pharmaceutically acceptable salts thereof. 8. A compound according to claim 7 wherein Y is hydrogen and Z is said compound being a member selected from the group consisting of nitrosoeveminomicin D, hydroxylaminoeverninomicin D, nitrone derivatives of hydroxylaminoeverninomicin D, and pharmaceutically acceptable salts thereof.

9. A compound of claim 8 wherein X is said compound identified as hydroxylaminoeverninomicin D, having the following physical characteristics: an R,=0.45 on silica gel plates (2000 p thick) when using 50% acetone in benzene as developing solvent; the following elemental analysis: C=5l.8l%, H=6.64% N=0.59%, and Cl=4.55%; a specific rotation of about -39.3 as measured in chloroform at 1% concentration by the D-line of sodium at 26C; a melting point of about l65l68C when measured on a Kofler block; has ultraviolet absorption maxima at 215 my and 292 mg. with e 1 cm. in methanol equal to about 19.2 and 3.4, respectively, has an infrared spectrum when dissolved in chloroform substantially as shown in FIG. 1; has a nuclear magnetic resonance spectrum in deuterochloroform substantially as shown in FIG. 2; has an antibacterial spectrum including the bacteria enumeratedin Table l.

10. A compound of claim 8 wherein X is N=O, said compound identified as nitrosoeveminomicin D, having the following physical characteristics: an R about 0.78 on silica gel plates (2000 p.) when using acetone- /ethyl acetate/benzene (2:2:1) as developing solvent; the following elemental analysis: C=51.66%, H=6.5 2%, N=O.84% and Cl=4.50%; a specific rotation of -30.8 as measured in chloroform at 1% concentration by the D- line of sodium at about 26C; a melting point of about l63l65C when measured on a Kofler block; has ultraviolet absorption maxima at 212 my. and 292 mp. with e 1 cm. in methanol=to 16.05 and 5.60, respectively; has an infrared spectrum when dissolved in chloroform substantially as shown in FIG. 3; has a nuclear magnetic resonance spectrum substantially as shown in FIG. 4; has an antibacterial spectrum including the bacteria enumerated in Table l.

11. A compound of claim 8 which is the sodium salt of a compound of the structural formula therein wherein X is said compound being hydroxylaminoeverninomicin D sodium salt.

12. A compound of claim 8 which is the N-methylglucamine salt of a compound of the structural formula wherein X is said compound being hydroxylaminoeveminomicin D N-methylglucamine salt.

13. A compound according to claim 7 wherein Y is hydroxyl and Z is said compound being a member selected from the group consisting of nitrosoeveminomicin B, hydroxylaminoeverninomicin B, nitrone derivatives of hydroxylaminoeverninomicin B, and the pharmaceutically acceptable salts thereof.

14. A compound of claim 13 wherein X is identified as hydroxylaminoeverninomicin B, having the following physical characteristics: an R,= 0.18 on ing the bacteria enumerated in Table l.

15. A compound of claim 7 wherein Y and Z are each hydrogen, said compound being a member selected from the group consisting of nitrosoeveminomicin C, hydroxylaminoeveminomicin C, nitrone derivatives of hydroxylaminoeveminomicin C, and the pharmaceutically acceptable salts thereof.

16. A compound of claim 15 wherein X is identified as hydroxylaminoeverninomicin C, having the following physical characteristics: an R,= to 0.27 on silica gel preparative plates (2000 p. thick) when using acetone in benzene as developing solvent; the following elemental analysis: C=5l.35, 51.74%; H=6.66, 6.75%; N=0.93, 1.09% and Cl=5.75%; a specific rotation of about --2l.6 as measured in methanol at 1% concentration by the D-line of sodium at 26C; is

ima when dissolved in chloroform substantially as shown in FIG. 9; has melting point of about -168C when measured on a Kofler block; has an antibacterial spectrum including the bacteria enumerated in Table l.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION- PATENT NO. ,915,956 I DATED October 28, 1975 INVENTOR(S) Ashit K. Ganguly et al It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 5, line 51, "The isolation and purification and everninomicins B" should read --The isolation and purification of everninomicins B--. Column i, line 16, "nitrosoeverniomicin D; in" should read --nitrosoeverninomicin D; in--. Column 10, line L0, "the sodium slt" should read --the sodium salt--. Column 10, line 67, "Pancreatic digest or casein" should read Pancreatic digest of casein--. Column 11, line +5, "doses in rats for" should read --doses in mice for--. Column 11, lines 62 and 65, or milligrams" should read --l0 or 25 mg/kg--. Column 1 line 17, "water, ethantol" should read --water, ethanol--. Column 15, line 59, "magnetic rsonance" should read --magnetic resonance--. Column 16, line ME, "My, l 25;" should read -M 1, a: 1 i;- Column 17, line 8, -65. 2" should -2 read A 3 4 -65. 2 Column 17, line 58, "L 6 +6. 0 hould read "[04:6 i6. 0 Column 22, lines 28 and 29,

"hydroxylaminoeverninomicin D, hydroxylaminoeverninomicin D," should read -hydroxylaminoeverninomicin C, hydroxylaminoeverninomicin D,--. Claim 5, Column 2h, line 55, '"ninomicr'un b," should read --ninomicin B,

Signed and Scaled this" Twentieth Day Of July 1976 [SEAL] A ttes t:

RUTH C. MASON Arresting Officer C. MARSHALL DANN Commissioner oj'Patenrs and Trademarks 

1. THE PROCESS WHICH COMPRISES TREATING AN EVERNINOMICIN ANTIBIOTIC SELECTED FROM THE GROUP CONSSTING OF EVERNINOMICIN B, EVERNINOMICIN C, AND EVERMINOMICIN D WITH ALUMINUM AMALGAM IN AQUEOUS LOWER ALKANOL AT ROOM TEMPERATURE WHEREBY IS PRODUCED A PRODUCT COMPRISING A MEMBER SELECTED FROM THE GROUP CONSISTING OF THE CORRESPONDING HYDROXYLAMINOEVERNINOMICIN, THE CORRESPONDING NITROSOEVERNINOMICALCIN, AND MIXTURES THEREOF.
 2. The process according to claim 1 including the added step of isolating the hydroxylaminoeverninomicin derivative thereby produced.
 3. The process according to claim 1 wherein said aluminum amalgam is an amalgam of Reynolds Wrap aluminum foil.
 4. The process according to claim 1 wherein said aluminum amalgam is an amalgam of Reynolds Wrap aluminum foil and wherein said aqueous lower alkanol is 95% aqueous ethanol.
 5. The process for the preparation of nitrosoeverninomicin selected from the group consisting of nitrosoeverninomicin B, nitrosoeverninomicin C, and nitrosoeverninomicin D which comprises treating the corresponding hydroxylaminoeverninomicin selected from the group consisting of hydroxylaminoeverninomicin b, hydroxylaminoeverninomicin C, and hydroxylaminoeverninomicin D with an oxidizing reagent selected from the group consisting of aerial oxidation in an alkaline solution, and an alkali metal hypobromite in an aprotic solvent.
 6. The process according to claim 5 wherein said oxidizing reagent is sodium hypobromite in tetrahydrofuran.
 7. A compound selected from the group consisting of nitrosoeverninomicin B, nitrosoeverninomicin C, nitrosoeverninomicin D, hydroxylaminoeverninomicin B, hydroxylaminoeverninomicin C, hydroxylaminoeverninomicin D, nitrone derivatives of said hydroxylaminoeverninomicins B, C, and D, said compound having the following structural formula:
 8. A compound according to claim 7 wherein Y is hydrogen and Z is
 9. A compound of claim 8 wherein X is
 10. A compound of claim 8 wherein X is -N O, said compound identified as nitrosoeverninomicin D, having the following physical characteristics: an Rf about 0.78 on silica gel plates (2000 Mu ) when using acetone/ethyl acetate/benzene (2:2:1) as developing solvent; the following elemental analysis: C 51.66%, H 6.52%, N 0.84% and Cl 4.50%; a specific rotation of -30.8 as measured in chloroform at 1% concentration by the D-line of sodium at about 26*C; a melting point of about 163*-165*C when measured on a Kofler block; has ultraviolet absorption maxima at 212 m Mu and 292 m Mu with epsilon 1 % 1 cm. in methanol to 16.05 and 5.60, respectively; has an infrared spectrum when dissolved in chloroform substantially as shown in FIG. 3; has a nuclear magnetic resonance spectrum substantially as shown in FIG. 4; has an antibacterial spectrum including the bacteria enumerated in Table
 1. 11. A compound of claim 8 which is the sodium salt of a compound of the structural formula therein wherein X is
 12. A compound of claim 8 which is the N-methylglucamine salt of a compound of the structural formula wherein X is
 13. A compound according to claim 7 wherein Y is hydroxyl and Z is
 14. A compound of claim 13 wherein X is
 15. A compound of claim 7 wherein Y and Z are each hydrogen, said compound being a member selected from the group consisting of nitrosoeverninomicin C, hydroxylaminoeverninomicin C, nitrone derivatives of hydroxylaminoeverninomicin C, and the pharmaceutically acceptable salts thereof.
 16. A compound of claim 15 wherein X is 